Our long-term goal is to understand the fundamental mechanisms underlying the physiological and pathological roles of K+ channels and to develop new approaches to control their activity for treating diseases such as autoimmune diseases and cardiac arrhythmia. Here, pursuing an on-going systematic investigation, we will help establish a new conceptual framework to better rationalize the voltage-gating mechanism in voltage-gated K+ (Kv) channels and to delineate critical regions that are suitable targets for modulation of Kv channel activity. In specific Aim#1 of the coming grant cycle, we will genetically engineer a Kv channel with a minimal voltage-dependent controller that can not only sense voltage but also couple the sensor to the channel gate. By doing so, we will delineate the regions of the channel protein that are essential for conferring voltage sensing upon a Kv channel. These regions may then represent key sites that can be targeted to modulate Kv channel activity. In Aim#2, we will examine the significance of an unexpected structural feature of the voltage sensor. Positively charged residues in the N-terminal part of the fourth transmembrane segment (NTS4) in the Kv channel protein sense membrane voltage. Traditionally, it had been assumed that upon a voltage change, S4 moves with respect to the rest of the channel protein. Unexpectedly, in all crystal structures of Kv channels to date, NTS4, the C-terminal part of S3 (S3b) and their linker form a paddle-like, helix-turn-helix motif. The observation of this unexpected paddle motif led to the paddle model which views the motif as a rigid functional unit that moves in response to voltage changes. Thus far, there is little experimental information regarding the role of S3b in voltage gating. We will try to uncover the significance of the paddle motif in voltage gating with four series of conceptually innovative and complementary experiments. We will carry out all the studies with combined biophysical, biochemical and molecular biological techniques. The success of our studies will (i) advance our understanding of the voltage-gating mechanism by establishing a new conceptual framework, (ii) help unify the current competing models accounting for the voltage-sensing mechanism, and (iii) delineate key sites that can be exploited to develop drug therapies in the future. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to establish evidence for a new conceptual framework for better rationalizing the voltage-gating mechanism, and to delineate the regions that are functionally essential for voltage gating. These regions could then be exploited as drug targets in treating certain diseases such as autoimmune diseases and cardiac arrhythmia.